PROJECT SUMMARY Hypercalciuria occurs in 5-10% of the population and is the most common cause of calcium (Ca) kidney stone disease. Ca reabsorption takes place in different nephron segments; however, evidence from human subjects indicates that proximal tubule Ca reabsorption is decreased in stone formers. Furthermore, studies in both animals and humans suggest that increased proximal tubule Ca reabsorption accounts for the hypocalciuric effect of thiazide diuretics, the first line medication for hypercalciuria. Compared to Ca reabsorption in the distal tubule, much less is known about the mechanism of Ca reabsorption in the proximal tubule. Lines of evidence from classical physiological studies indicate the existence of an active Ca transport pathway in the proximal straight tubule, the last portion of the proximal tubule where Ca can be reabsorbed before tubular fluid enters the loop of Henle where supersaturation of Ca phosphate may occur due to water extraction. Active Ca reabsorption in this segment of the nephron may remove Ca in the tubular fluid to a level below that in the plasma. The objective of this proposal is to elucidate the roles of a novel active Ca transport pathway in the proximal straight tubule in causing and treating hypercalciuria. The central hypothesis is that active Ca reabsorption in the proximal tubule is disrupted by postprandial insulin surge and under hyperinsulinemia and is enhanced by thiazides. In addition, a Ca channel in the apical side of proximal straight tubule may act as a Ca sensor to regulate overall Ca homeostasis. It has been shown that postprandial insulin secretion and Ca excretion are both increased in hypercalciuric stone formers. Our preliminary studies indicate that TRPV6, a key Ca channel in the transcellular Ca transport pathway, is distributed to the proximal straight tubule, and TRPV6 activity is inhibited by insulin, and the hypocalciuric effect of hydrochlorothiazide is significantly reduced in TRPV6 null mice. These observations support our hypothesis, which implies that inhibition of TRPV6 by insulin contributes to postprandial hypercalciuria and hypercalciuria due to compensatory hyperinsulinemia as a result of insulin resistance. We will test our hypothesis and achieve our research objective by pursuing the following specific aims. 1. Determine the inhibitory effect of insulin on active Ca reabsorption in the proximal tubule. 2. Determine to what extent TRPV6 mediates the hypocalciuric effect of thiazides. 3. Determine the Ca-sensor roles of TRPV6 in the proximal straight tubule that regulate vitamin D synthesis and paracellular Ca transport. Approaches ranging from Ca influx in human proximal tubule cells to metabolic studies using TRPV6 null mice will be employed to accomplish our research goals. Upon successful completion of the proposed research, we expect to unveil the molecular mechanism for the active Ca reabsorption in the proximal tubule that regulates Ca reabsorption and Ca homeostasis. The inhibition of the active Ca reabsorption by insulin provides a molecular basis for the postprandial hypercalciuria in many stone formers and elevated Ca excretion in prediabetes. These outcomes will help to develop a new strategy to prevent hypercalciuria and kidney stone disease.